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Article

Upcycled Postbiotic Cell-Free Supernatants from Limosilactobacillus fermentum MG901 and MG4237 Alleviated Oxidative Stress-Induced Dysfunction in Human Follicle Dermal Papilla Cells

by
Chae Young Jeon
1,
Ji Yeon Lee
2,
Jungwon Min
1,
Jeong-Yong Park
2,
Minha Kim
1,
Wonchan Yoon
2,
Soo-Im Choi
2 and
Dong Wook Shin
1,*
1
Research Institute for Biomedical and Health Science, Konkuk University, Chungju 27478, Republic of Korea
2
MEDIOGEN Co., Ltd., Biovalley 1-ro, Jecheon-si 27159, Republic of Korea
*
Author to whom correspondence should be addressed.
Cosmetics 2026, 13(1), 46; https://doi.org/10.3390/cosmetics13010046
Submission received: 2 January 2026 / Revised: 7 February 2026 / Accepted: 8 February 2026 / Published: 18 February 2026
(This article belongs to the Special Issue Skin Care and Innovation from the Perspective of Molecular Dermatology)
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Abstract

Oxidative stress–induced dysfunction of hair follicle dermal papilla cells (HFDPCs) is a key factor in the progression of hair loss. In this study, upcycled postbiotic cell-free supernatants (CFSs), derived from Limosilactobacillus fermentum (L. fermentum) MG901 and MG4237, which are typically discarded after fermentation, were evaluated for their protective effects in H2O2-damaged human dermal papilla cells. The CFS exhibited no cytotoxicity and significantly enhanced wound-healing capacity while suppressing intracellular reactive oxygen species accumulation under oxidative stress conditions. In addition, treatment with CFS restored mitochondrial function, indicating recovery from H2O2-induced cellular damage. Dermal papilla cell-specific functional markers, including alkaline phosphatase activity, were also significantly increased following treatment. Mechanistic analyses further revealed that these protective effects were associated with modulation of Wnt/β-catenin signaling as well as regulation of mitochondrial function. Collectively, these findings suggest that upcycled postbiotic CFS from L. fermentum MG901 and MG4237 mitigates oxidative stress-induced dermal papilla cell dysfunction, supporting its potential application as a sustainable cosmetic ingredient for alleviating hair loss.

1. Introduction

Hair loss is a complex biological phenomenon driven by genetic, hormonal, and environmental factors, among which oxidative stress is recognized as a critical contributor to follicular dysfunction [1,2]. Excessive accumulation of reactive oxygen species (ROS) disrupts cellular homeostasis within the hair follicle microenvironment, leading to impaired proliferation, metabolic imbalance [3], and premature functional decline of hair follicle dermal papilla cells (HFDPCs) [4,5]. Because dermal papilla cells serve as key regulators of hair cycle progression and follicular inductivity [6], oxidative stress–induced damage to these cells is closely associated with the onset and progression of hair thinning [7].
Mitochondrial dysfunction is a central event in oxidative stress–mediated dermal papilla impairment [8]. Excessive ROS disrupts mitochondrial activity and energy metabolism and impairs mitochondrial quality control, including mitophagy [9,10]. In parallel, dysregulation of growth-associated signaling pathways, particularly the Wnt/β-catenin pathway [11], further attenuates dermal papilla cell function and hair growth–related signaling [12,13]. The Wnt/β-catenin pathway plays a pivotal role in maintaining dermal papilla inductivity by regulating cell proliferation and transcription of hair growth–associated genes [14]. Suppression of β-catenin signaling has been closely linked to reduced anagen maintenance and follicular miniaturization, whereas its activation promotes hair follicle regeneration [15]. Given its established role in regulating dermal papilla cell inductivity and maintaining the anagen phase of the hair cycle, the Wnt/β-catenin pathway was selected as a key molecular target for mechanistic analysis in this study [16,17]. Oxidative stress is known to impair dermal papilla cell function by disrupting intracellular signaling balance, and alterations in β-catenin stability represent a critical molecular event linking cellular stress to functional decline [18]. Therefore, focusing on Wnt/β-catenin–related signaling provides a biologically relevant framework for investigating oxidative stress–induced dysfunction and its modulation by postbiotic-derived CFS.
In addition to biological considerations, sustainability has emerged as an important paradigm in cosmetic ingredient development [19,20]. Lactic acid bacteria (LAB) are extensively utilized in the food and biotechnology industries [21], generating large quantities of fermentation-derived cell-free supernatants (CFSs) that are typically discarded after microbial biomass collection [22,23]. These culture supernatants contain diverse metabolites, including organic acids, peptides, and low-molecular-weight compounds, collectively described as postbiotics [24]. Postbiotics have attracted attention for cosmetic applications due to their chemical stability and potential bioactivity [25]. However, their effects on hair follicle-related cells and oxidative stress–associated hair loss mechanisms remain insufficiently explored [26,27].
LAB produces a diverse range of metabolites during fermentation, including organic acids and other low-molecular-weight compounds that can exert biological effects on host cells [28]. These bioactive compounds include hydrogen peroxide, organic acids, and various antifungal metabolites, including phenyl lactate, propionate, and hydroxyphenyl lactate [29]. Among LAB species, Limosilactobacillus fermentum (L. fermentum) is widely used as a reference strain in comparative studies due to its high metabolic activity and ability to produce diverse bioactive compounds during fermentation [30]. In particular, the CFS of L. fermentum, which contains acetate and lactate, has been reported to exhibit anti-inflammatory and antioxidant properties [31]. L. fermentum has also been shown to exert multiple skin-related beneficial effects, including antioxidant, anti-inflammatory, and skin barrier–protective activities, highlighting its potential relevance in skin health-associated applications [32]. Despite these findings, the biological activity of LAB–derived CFS can vary substantially depending on strain-specific fermentation characteristics and metabolic profiles, and the effects of L. fermentum-derived CFS on hair follicle dermal papilla cells (HFDPCs) remain poorly understood. Therefore, this study aimed to evaluate the antioxidant and cytoprotective effects of CFS derived from L. fermentum in HFDPCs under oxidative stress conditions.
In this study, upcycled postbiotic CFS, previously treated as an industrial by-product, was repurposed as candidate cosmetic ingredients. Their biological efficacy was evaluated using H2O2-damaged HFDPCs as an in vitro model of oxidative stress–induced hair loss. Cell viability, wound-healing capacity, intracellular ROS levels, and dermal papilla-specific functional markers, including alkaline phosphatase activity, were assessed to determine whether CFS restored cellular physiological activity. Furthermore, mechanistic analyses were conducted to investigate the involvement of Wnt/β-catenin signaling and mitochondrial function. Through these investigations, this study aimed to establish scientific evidence supporting L. fermentum-derived CFS as a postbiotic-based cosmetic ingredient for alleviating hair loss and to provide a foundation for the development of sustainable functional cosmetic materials.

2. Materials and Methods

2.1. Sample Preparation

L. fermentum MG901 and MG4237 were isolated from vaginal samples obtained from healthy women (Table 1). Each strain was cultured in Man Rogosa Sharpe broth (Difco, Sparks, MD, USA) at 37 °C for 18 h. After cultivation, bacterial cells were removed by centrifugation at 1500× g for 10 min, and the CFS was collected and filtered through a 0.22 µm polytetrafluoroethylene membrane filter (ADVANTEC, Tokyo, Japan) and stored under frozen conditions. The CFS was used for subsequent in vitro experiments.

2.2. Metabolite Analysis of L. fermentum-Derived CFS

The CFS of L. fermentum MG901 and MG4237 was analyzed to determine residual glucose and organic acid (lactate and acetate) concentrations. The CFS samples, prepared as described above, were subjected to centrifugation to ensure complete removal of bacterial cells. To prevent pH-dependent artifacts in the cellular assays, the pH of the CFS was adjusted to 7.4 using 1 M NaOH. The neutralized supernatants were subsequently filtered through a 0.22 µm polytetrafluoroethylene membrane filter (ADVANTEC, Tokyo, Japan) before analysis. Glucose, lactate, and acetate concentrations were quantified using a Roche Cedex® Bio Analyzer (06395554001, Roche Diagnostics, Laval, QC, Canada). For calibration and quantification, assay kits for glucose (06343732001), lactate (06343759001), and acetate (07395442001) (Roche Diagnostics) were used in accordance with the manufacturer’s protocols. Briefly, enzymatic assays specific to each analyte were applied, and the instrument automatically calculated concentrations based on absorbance measurements.

2.3. Cell Culture

Human follicle dermal papilla cells (HFDPCs) obtained from PromoCell (Heidelberg, Germany) were maintained in dermal papilla cell growth medium supplemented with a proprietary growth factor mixture and 1% penicillin–streptomycin. Cells were cultured at 37 °C in a humidified incubator containing 5% CO2. For routine maintenance, commercially available ready-to-use HFDPC medium and a Detach kit (PromoCell) were employed. The Detach kit, consisting of HEPES-buffered balanced salt solution, trypsin/EDTA, and a trypsin neutralization solution, was used to facilitate gentle cell dissociation. Cells were passaged when they reached approximately 80–90% confluence and reseeded into 75 mm culture flasks, typically at 3-day intervals. In this study, HFDPCs were used between passages 5 and 9 to ensure experimental consistency.

2.4. Cell Viability

Cell viability was evaluated using the EZ-Cytox assay kit (DoGenBio, Seoul, Republic of Korea). HFDPCs were seeded in 96-well plates and allowed to stabilize for 24 h before treatment. Cells were then treated with the CFS of L. fermentum MG901 or MG4237 conditioned medium at the indicated concentrations (0.5–2%) for an additional 24 h. Following treatment, EZ-CytoX reagent was added to each well and incubated at 37 °C according to the manufacturer’s instructions. Absorbance was measured at 450 nm using a microplate reader (BioTek, Winooski, VT, USA).

2.5. Wound-Healing Assay

HFDPCs were seeded in 6-well plates and grown to approximately 80% confluence. A straight scratch was made across the cell monolayer using a sterile 1000 µL pipette tip (Axygen Scientific Inc., Union City, CA, USA), and floating cells were removed by gently washing with Dulbecco’s Phosphate-Buffered Saline (DPBS). The cells were then incubated with fresh medium containing the CFS of L. fermentum MG901 or MG4237 at the indicated concentrations for 24 h. After CFS treatment, oxidative stress was induced by exposure to H2O2 (200 µM) for 2 h. Wound closure was evaluated by acquiring phase-contrast images at 0 h and 24 h after scratch formation using a phase-contrast microscope (ECLIPSE Ts2, Nikon, Tokyo, Japan) and analyzed with Fiji ImageJ software (version 1.53e; National Institutes of Health, Bethesda, MD, USA).

2.6. Alkaline Phosphatase Staining(ALP) Assay

HFDPCs were seeded in 24-well plates and allowed to adhere under standard culture conditions. Cells were pre-treated for a total of 24 h, including exposure to H2O2 (200 µM) for 2 h to induce oxidative stress, followed by treatment with the CFS of L. fermentum MG901 or MG4237 at the indicated concentrations. After treatment, cells were briefly fixed and washed with Phosphate-Buffered Saline with Tween 20 (PBST). Alkaline phosphatase staining was performed using an AP staining solution according to the manufacturer’s instructions under light-protected conditions. Stained cells were rinsed with DPBS and visualized using a light microscope (ECLIPSE Ts2, Nikon, Tokyo, Japan). ALP-positive staining intensity was quantified using Fiji ImageJ software (version 1.53e).

2.7. Measurement of Intracellular ROS

HFDPCs were seeded in confocal dishes and cultured for 24 h before treatment. Cells were pre-treated for a total of 24 h, including H2O2 (200 µM) exposure for 2 h, followed by treatment with the CFS of L. fermentum MG901 or MG4237 at the indicated concentrations. After treatment, cells were washed with DPBS and incubated with 2,7-Dichlorofluorescin diacetate (DCF-DA) solution (final concentration, 10 µM) for 20 min at 37 °C in the dark. Cells were then rinsed with DPBS, and fluorescence images were acquired using a live-cell fluorescence microscope (ECLIPSE Ti2, Nikon, Tokyo, Japan). Intracellular ROS levels were quantified using Fiji ImageJ software (version 1.53e).

2.8. Measurement of Membrane Potential in Mitochondria

HFDPCs were plated in confocal dishes and incubated for 24 h. Cells were pre-treated for 24 h, including H2O2 (200 µM) exposure for 2 h, followed by treatment with the CFS of L. fermentum MG901 or MG4237. After treatment, cells were washed with DPBS and stained with JC-1 solution (final concentration, 10 µM) for 15 min at 37 °C. Following staining, cells were rinsed with DPBS, and fluorescence images were obtained using a live-cell imaging microscope (ECLIPSE Ti2, Nikon, Tokyo, Japan). Mitochondrial membrane potential was assessed by calculating the red-to-green fluorescence ratio using Fiji ImageJ software (version 1.53e).

2.9. Western Blot Assay

HFDPCs were seeded in culture dishes and incubated for 24 h before treatment. Cells were pre-treated for a total of 24 h, including exposure to H2O2 (200 µM) for 2 h, followed by treatment with the CFS of L. fermentum MG901 or MG4237. After treatment, cells were washed with DPBS and lysed using RIPA buffer (Thermo Fisher Scientific, Waltham, MA, USA) to extract total protein. Cell lysates were clarified by centrifugation, and protein concentrations were determined using a bicinchoninic acid (BCA) protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA). Equal amounts of protein were denatured, separated by SDS–PAGE, and transferred onto membranes. Membranes were blocked with 5% non-fat milk in Tris-buffered saline containing 0.1% Tween 20 (TBS-T) and incubated overnight at 4 °C with primary antibodies against β-catenin, phosphorylated GSK3β at Ser9 (p-GSK3β Ser9), total GSK3β, and β-actin (Cell Signaling Technology, Danvers, MA, USA) at a dilution of 1:1000. Membranes were then incubated with appropriate horseradish peroxidase–conjugated secondary antibodies at a dilution of 1:5000. Protein bands were visualized using an enhanced chemiluminescence reagent and captured with an iBright 1500 imaging system (Thermo Fisher Scientific). Band intensities were quantified using Fiji ImageJ software (version 1.53e).

2.10. Statistical Analysis

All data were derived from at least three independent experiments and are expressed as the mean ± standard deviation. Statistical analyses were conducted using GraphPad Prism software (version 8.0.1; GraphPad Software, San Diego, CA, USA). Statistical significance was determined using Student’s t-test for comparisons between two groups and one-way ANOVA followed by Tukey’s multiple comparison test for comparisons among three or more groups. All data are presented as the mean ± standard deviation (SD), and a p-value < 0.05 was considered statistically significant.

3. Results

3.1. Metabolic Profiles of L. fermentum MG901- and MG4237-Derived CFS

To characterize the metabolic profiles of the CFS derived from L. fermentum MG901 and MG4237, residual glucose, lactate, and acetate levels were measured using a Cedex Bio Analyzer (Table 2). The unfermented basal medium contained a high concentration of glucose (27,022.45 ± 521.08 mg/L), along with detectable levels of lactate (4222.96 ± 46.78 mg/L) and acetate (6.01 ± 0.02 mM). Following fermentation and removal of bacterial cells, both L. fermentum MG901- and MG4237-derived CFS exhibited a marked reduction in residual glucose levels compared with the basal medium (Table 2). The residual glucose level in the L. fermentum MG901-derived CFS was reduced to 8172.66 ± 344.71 mg/L, whereas the L. fermentum MG4237-derived CFS exhibited a further decrease to 7015.37 ± 342.28 mg/L. In contrast, the lactate level in both L. fermentum MG901- and MG4237-derived CFS remained comparable to those observed in the basal medium, measuring 4199.01 ± 65.22 mg/L and 4262.24 ± 72.04 mg/L, respectively. Acetate level in L. fermentum MG901-derived CFS was similar to the basal medium (6.09 ± 0.16 mM), whereas L. fermentum MG4237-derived CFS exhibited a higher acetate concentration (7.11 ± 0.28 mM).

3.2. Cell Viability of HFDPCs Treated with MG901- and MG4237-Derived CFS

To determine appropriate treatment concentrations for subsequent functional assays, the effects of CFS derived from L. fermentum MG901 and MG4237 on HFDPC viability were evaluated using the EZ-CytoX assay. As shown in Figure 1, MG901-derived CFS maintained cell viability at a level comparable to the control at 0.5% concentration, whereas a gradual decline was observed at higher concentrations. In contrast, MG4237-derived CFS exhibited stable cell viability across the tested range, with no marked reduction even at 2%. Importantly, none of the tested concentrations of either CFS induced overt cytotoxic effects, as cell viability remained close to baseline levels.
Based on these profiles, concentrations that preserved cellular integrity while ensuring sufficient exposure were selected for subsequent experiments. MG901-derived CFS was applied at 0.5%, corresponding to the highest concentration that maintained viability comparable to the control group, whereas MG4237-derived CFS was used at 2%, which showed sustained cell viability without detectable adverse effects. These concentrations were therefore considered suitable for further evaluation of hair loss-related functional and mechanistic outcomes in HFDPCs.

3.3. MG901- and MG4237-Derived CFS Enhanced Migration in H2O2-Damaged HFDPCs

Because the migratory capacity of dermal papilla cells is essential for maintaining follicular structure and supporting hair cycle–related remodeling, a wound-healing assay was performed to evaluate functional recovery under oxidative stress conditions [33,34]. HFDPCs were exposed to H2O2 (200 µM) to induce migration impairment, and wound closure was monitored.
As shown in Figure 2, H2O2 treatment markedly delayed gap closure compared with the control. Co-treatment with biotin (5 µg/mL), used as a positive control, resulted in partial recovery of wound closure [35]. Treatment with MG901-derived CFS (0.5%) or MG4237-derived CFS (2%) increased cell movement into the wounded area relative to the H2O2-treated group, leading to a visibly reduced gap after 24 h. By 48 h, near-complete closure across all groups saturated the assay, masking any sample-specific effects. These observations indicated that CFS derived from L. fermentum MG901 and MG4237 alleviated oxidative stress–induced suppression of dermal papilla cell migration.

3.4. MG901- and MG4237-Derived CFS Modulated Alkaline Phosphatase Activity in H2O2-Damaged HFDPCs

Alkaline phosphatase (ALP) activity was examined as a functional indicator of dermal papilla cell inductivity, which is closely associated with hair growth–related signaling and follicular maintenance [36,37]. Because oxidative stress is known to suppress ALP expression in dermal papilla cells, ALP staining and quantitative analysis were performed to evaluate whether CFS derived from L. fermentum restored dermal papilla–specific functionality under oxidative conditions.
As shown in Figure 3, exposure to H2O2 (200 µM) markedly reduced ALP staining intensity and enzymatic activity compared with the control. Co-treatment with biotin (5 µg/mL) increased ALP activity relative to the H2O2-treated group. Treatment with MG901-derived CFS (0.5%) resulted in a clear enhancement of ALP staining and activity in H2O2-damaged cells. Similarly, MG4237-derived CFS (2%) increased ALP activity, showing a pronounced elevation compared with the oxidative stress group. Quantitative analysis confirmed that both CFSs significantly increased ALP activity under H2O2-induced stress conditions.

3.5. MG901- and MG4237-Derived CFS Reduced Intracellular ROS Levels in H2O2-Damaged HFDPCs

Intracellular ROS levels were assessed using the DCF-DA assay to determine whether CFS derived from L. fermentum modulated oxidative stress in dermal papilla cells. Because excessive ROS accumulation is a primary driver of oxidative damage–induced functional impairment in HFDPCs, DCF-DA fluorescence was measured as an index of cellular status under H2O2-induced stress conditions.
As shown in Figure 4, exposure to H2O2 (200 µM) resulted in a marked increase in DCF-DA fluorescence compared with the control, indicating elevated intracellular ROS generation. Co-treatment with biotin (5 µg/mL), used as a positive control, substantially reduced fluorescence intensity relative to the H2O2-treated group. Treatment with MG901-derived CFS (0.5%) noticeably attenuated DCF-DA fluorescence in H2O2-damaged cells. Similarly, MG4237-derived CFS (2%) markedly decreased fluorescence intensity compared with cells treated with H2O2 alone. Quantitative analysis confirmed that both CFSs significantly suppressed intracellular ROS accumulation under oxidative stress conditions.

3.6. MG901 and MG4237 Modulated Mitochondrial Membrane Potential in H2O2-Damaged HFDPCs

Mitochondrial membrane potential (ΔΨm) was evaluated using the JC-1 assay to assess mitochondrial integrity under oxidative stress [38,39]. Because loss of ΔΨm is an early indicator of mitochondrial dysfunction that precedes broader metabolic impairment in dermal papilla cells [40,41], JC-1 staining was employed to determine whether CFS derived from L. fermentum influenced mitochondrial stability following H2O2 exposure.
As shown in Figure 5, treatment with H2O2 (200 µM) markedly decreased the JC-1 aggregate/monomer fluorescence ratio compared with the control, reflecting disruption of mitochondrial membrane potential. Co-treatment with biotin (5 µg/mL), used as a positive control, increased the JC-1 ratio relative to the H2O2-treated group. Treatment with MG901-derived CFS (0.5%) resulted in an elevated JC-1 aggregate/monomer ratio in H2O2-damaged cells. Similarly, MG4237-derived CFS (2%) increased the JC-1 ratio compared with oxidative stress alone. Quantitative analysis confirmed that both CFSs significantly improved the JC-1 fluorescence ratio under H2O2-induced stress conditions.

3.7. MG901- and MG4237-Derived CFS Activated Wnt/β-Catenin Signaling in H2O2-Damaged HFDPCs

Protein expression associated with Wnt/β-catenin signaling was analyzed by Western blotting to examine molecular events underlying functional recovery in dermal papilla cells [42]. Because activation of the Wnt/β-catenin pathway is essential for maintaining dermal papilla inductivity and hair growth–related signaling, β-catenin accumulation and phosphorylation status of glycogen synthase kinase-3β (GSK3β) were evaluated under oxidative stress conditions [43,44].
As shown in Figure 6, exposure to H2O2 (200 µM) reduced β-catenin protein levels compared with the control, accompanied by a decrease in phosphorylated GSK3β at Ser9 (p-GSK3β Ser9) relative to total GSK3β. Co-treatment with biotin (5 µg/mL) increased β-catenin expression and restored p-GSK3β levels under oxidative stress. Treatment with MG901-derived CFS (0.5%) elevated β-catenin protein abundance compared with the H2O2-treated group. Similarly, MG4237-derived CFS (2%) increased β-catenin levels and enhanced the p-GSK3β/total GSK3β ratio. Densitometric analysis confirmed that both CFSs modulated key components of Wnt/β-catenin signaling in H2O2-damaged dermal papilla cells.

4. Discussion

Oxidative stress is recognized as one of the critical factors contributing to hair follicle dysfunction and hair loss progression, acting in concert with genetic, hormonal, and environmental influences [45,46]. In this study, H2O2-induced oxidative damage was employed as an in vitro model to simulate stress-related dysfunction in human dermal papilla cells, allowing systematic evaluation of the biological effects of L. fermentum-derived CFS from MG901 and MG4237. The findings collectively demonstrated that both CFSs alleviated oxidative stress–associated cellular impairment and restored multiple hair growth–related functional parameters.
Initial cytocompatibility assessment confirmed that MG901- and MG4237-derived CFS did not exert cytotoxic effects within the selected concentration ranges, ensuring that subsequent functional changes were not attributable to altered cell viability. Oxidative stress has been shown to impair dermal papilla cell function primarily by inducing cellular senescence and dysregulating hair growth–related signaling pathways, thereby compromising follicular maintenance and regeneration [47,48]. Treatment with MG901- or MG4237-derived CFS partially restored migratory capacity, suggesting recovery of cellular behavior essential for maintaining dermal papilla architecture and follicular integrity during the hair cycle [49].
Intracellular redox imbalance represents a primary trigger for oxidative stress–mediated cellular dysfunction [50,51]. In the present study, hydrogen peroxide exposure resulted in excessive accumulation of reactive oxygen species, whereas both CFSs significantly attenuated intracellular ROS levels. This reduction in oxidative burden was accompanied by improvements in mitochondrial membrane potential, as assessed by JC-1 staining. Given that mitochondrial depolarization is an early hallmark of oxidative damage and precedes broader metabolic failure [52], the observed restoration of mitochondrial membrane potential indicates that MG901- and MG4237-derived CFS supported mitochondrial stability under stress conditions.
Beyond general metabolic recovery, dermal papilla–specific functionality was further evaluated using alkaline phosphatase activity, a well-established marker of dermal papilla inductivity and hair growth potential [53,54]. Oxidative stress markedly reduced ALP activity, whereas treatment with CFS derived from MG901- or MG4237 restored ALP levels, indicating preservation of dermal papilla cell identity and functional competence. These findings suggest that the effects of the CFS extended beyond antioxidant protection to include recovery of lineage-specific cellular properties.
At the molecular level, disruption of Wnt/β-catenin signaling has been closely linked to oxidative stress–induced impairment of dermal papilla function and hair growth regulation [55,56]. Consistent with this concept, hydrogen peroxide treatment reduced β-catenin protein levels and altered the phosphorylation status of GSK3β. Treatment with MG901- or MG4237-derived CFS increased β-catenin abundance and enhanced the p-GSK3β/total GSK3β ratio, indicating reactivation of Wnt/β-catenin signaling under oxidative conditions (Figure 7). Although direct transcriptional targets were not examined in this study, restoration of this pathway aligns with the observed recovery of functional markers associated with hair growth.
L. fermentum is an obligate heterofermentative bacterium. Unlike homofermentative lactic acid bacteria, which convert glucose almost exclusively into lactate, heterofermentative strains utilize the phosphoketolase pathway. This pathway yields a variety of end-products, including lactate, ethanol, acetate, and CO2, depending on the redox balance and environmental conditions [57]. L. fermentum exhibits high metabolic adaptability, where the carbon flux can be diverted from lactate toward other metabolites such as acetate or ethanol to optimize energy yield or maintain cellular homeostasis [58]. In our data (Table 2), despite a significant decrease in glucose, stable lactate levels and a concurrent increase in acetate (particularly in L. fermentum MG4237) suggest a metabolic shift toward the acetate-producing branch of the heterofermentative pathway. Specifically, we observed a significant increase in acetate in MG4237-derived CFS. Acetate is a well-known short-chain fatty acid (SCFA) that can support mitochondrial function and potentially stabilize cellular signaling under stress [59]. This indicates that the consumed glucose was utilized for the production of alternative metabolites rather than additional lactate. The consistency of our results across triplicate experiments (n = 3), as reflected in the small standard deviations, further confirms that these metabolic profiles are characteristic of the specific strains and culture conditions used in this study.
The strain-specific metabolic profiles suggest that different bioactive components may drive the protective effects in HFDPCs. While the elevated acetate in MG4237-derived CFS likely contributes to mitochondrial stability and cellular signaling, the comparable effects observed with MG901—notwithstanding its basal levels of acetate—point toward the involvement of other functional elements. In the case of MG901, the bioactivity may be primarily mediated by a synergistic combination of exopolysaccharides (EPS), functional peptides, or other secreted factors. This multifaceted mode of action highlights that while both strains offer similar therapeutic outcomes, the underlying molecular drivers may differ.
Furthermore, the physiological relevance of these strains to the target application site merits consideration. Unlike lactic acid bacteria (LAB) strains commonly used in the food or biotechnology industries, which are primarily optimized for fermentation efficiency and industrial yield, human-derived strains have evolutionarily adapted to interact more effectively with human epithelial environments and maintain homeostasis [60,61].
Although the CFS was neutralized to pH 7.4 in this study to prevent pH-dependent artifacts during in vitro assays, the strains’ origin from a human microenvironment suggest a superior capacity to produced bioactive metabolites (postbiotics) that are physiologically compatible with human cells The host-adapted metabolic profiles likely include specific EPS and peptides that promote cellular stability and signaling, offering a strategic advantage for scalp-care applications compared to strains optimized for non-human industrial process. Consequently, the use of these host-adapted strains may provide a strategic advantage for practical applications aimed at promoting hair follicle health.
Beyond hair follicle-derived cells, postbiotics have been reported to exert protective and regulatory effects in various skin-associated cell types [62]. In epidermal keratinocytes, postbiotic preparations derived from lactic acid bacteria have been shown to attenuate oxidative stress, enhance barrier-related gene expression, and modulate inflammatory signaling. Similar antioxidant and cytoprotective properties have also been observed in dermal fibroblasts, where postbiotics contribute to cellular homeostasis under stress conditions [63,64]. In this context, the protective effects of MG901- and MG4237-derived CFS observed in HFDPCs are consistent with the broader biological activities reported for postbiotics in skin-related cellular systems.
Despite the promising in vitro findings, several limitations should be considered when interpreting the translational potential of postbiotic-derived CFS. From a biological perspective, further validation using ex vivo hair follicle cultures or in vivo systems is necessary to confirm efficacy within a complex tissue environment. From a regulatory standpoint, the translation of postbiotics into functional cosmetics or regenerative products faces significant hurdles. Regulatory authorities, such as the Food and Drug Administration (FDA), require rigorous safety assessments and, more importantly, strict batch-to-batch consistency and detailed characterization of the bioactive components within complex mixtures like CFS. Ensuring such quality control standards remain a critical challenge for the commercialization of novel biologically derived ingredients. These considerations highlight the need for further studies addressing both biological efficacy and regulatory feasibility.
In addition, although some LAB-derived culture supernatants are known to contain low levels of H2O2 as a metabolic by-product, its concentration in our CFS was not directly measured. Given that our CFS treatments lead to a significant decrease in intracellular ROS and enhanced mitochondrial stability against H2O2-induced damage, the protective bioactive metabolites within the CFS likely exerted a dominant effect over any endogenous peroxide. Nevertheless, future studies incorporating specific biochemical assays to measure peroxide content will be important for a more comprehensive characterization of the redox-related mechanisms of these postbiotic preparations.
Collectively, the present work provides mechanistic insight into the biological activity of LAB–derived culture media and supports their potential application as functional cosmetic ingredients targeting oxidative stress–related hair loss.

5. Conclusions

In this study, CFS derived from L. fermentum MG901 and MG4237 were shown to attenuate oxidative stress-associated functional impairment in HFDPCs under in vitro conditions. Treatment with MG901- and MG4237-derived CFS was associated with reduced intracellular ROS accumulation, partial restoration of mitochondrial membrane potential, and increased expression of dermal papilla-related functional markers. In addition, modulation of Wnt/β-catenin–related signaling was observed in H2O2-treated cells following CFS exposure.
Although these findings are limited to a cell-based oxidative stress model, they provide preliminary evidence supporting the potential utility of L. fermentum-derived CFS as a postbiotic ingredient for cosmetic applications targeting oxidative stress-related scalp and hair follicle dysfunction.

Author Contributions

Conceptualization, C.Y.J. and D.W.S.; formal analysis, C.Y.J., J.Y.L., J.M., J.-Y.P., M.K., W.Y. and S.-I.C.; funding acquisition, D.W.S.; investigation, C.Y.J., J.Y.L., J.M., J.-Y.P., M.K., W.Y. and S.-I.C.; methodology, C.Y.J.; writing—original draft, D.W.S.; writing—review and editing, D.W.S. All authors have read and agreed to the published version of the manuscript.

Funding

This research was supported by the Regional Innovation System & Education (RISE) program through the Chungbuk Regional Innovation System & Education Center, funded by the Ministry of Education (MOE) and the Chungcheongbuk-do, Republic of Korea (2025-RISE-11-003-03).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The data from this study are available upon demand from the corresponding author.

Conflicts of Interest

Authors Ji Yeon Lee, Jeong-Yong Park, Wonchan Yoon, Soo-Im Choi were employed by MEDIOGEN, Co., Ltd. The all authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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Figure 1. Effects of MG901- and MG4237-derived CFS on the viability of HFDPCs. Cell viability was assessed using the EZ-CytoX assay following treatment with MG901-derived CFS (0.5–2%) or MG4237-derived CFS (0.5–2%). Cell viability was expressed as a percentage relative to the control (Con). Data were presented as the mean ± SD of three independent experiments (n = 3).
Figure 1. Effects of MG901- and MG4237-derived CFS on the viability of HFDPCs. Cell viability was assessed using the EZ-CytoX assay following treatment with MG901-derived CFS (0.5–2%) or MG4237-derived CFS (0.5–2%). Cell viability was expressed as a percentage relative to the control (Con). Data were presented as the mean ± SD of three independent experiments (n = 3).
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Figure 2. Effects of MG901- and MG4237-derived CFS on Migration Capacity of H2O2-Damaged HFDPCs. (A) Representative phase-contrast images of wound closure in HFDPC monolayers at 0 and 24 h after scratch formation. Cells were exposed to H2O2 (200 µM) alone or co-treated with biotin (5 µg/mL), MG901-derived CFS (0.5%), or MG4237-derived CFS (2%). Blue lines indicate the wound margins. Scale bar = 50 µm. (B) Quantitative analysis of wound closure expressed as the percentage of gap closure at 24 h relative to the initial wound area. Data are presented as the mean ± SD of three independent experiments (n = 3). ## p < 0.01 vs. control; * p < 0.05 and ** p < 0.01 vs. H2O2-treated group.
Figure 2. Effects of MG901- and MG4237-derived CFS on Migration Capacity of H2O2-Damaged HFDPCs. (A) Representative phase-contrast images of wound closure in HFDPC monolayers at 0 and 24 h after scratch formation. Cells were exposed to H2O2 (200 µM) alone or co-treated with biotin (5 µg/mL), MG901-derived CFS (0.5%), or MG4237-derived CFS (2%). Blue lines indicate the wound margins. Scale bar = 50 µm. (B) Quantitative analysis of wound closure expressed as the percentage of gap closure at 24 h relative to the initial wound area. Data are presented as the mean ± SD of three independent experiments (n = 3). ## p < 0.01 vs. control; * p < 0.05 and ** p < 0.01 vs. H2O2-treated group.
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Figure 3. Effects of MG901- and MG4237-derived CFS on alkaline phosphatase activity in H2O2-damaged HFDPCs. (A) Representative images of ALP staining in control cells and cells exposed to H2O2 (200 µM) with or without biotin (5 µg/mL), MG901-derived CFS (0.5%), or MG4237-derived CFS (2%). (B) Quantification of ALP activity. Data were expressed relative to the control. Values represent the mean ± SD of three independent experiments (n = 3). ### p < 0.001 vs. control; *** p < 0.001 vs. H2O2-treated group. Scale bar = 20 µm.
Figure 3. Effects of MG901- and MG4237-derived CFS on alkaline phosphatase activity in H2O2-damaged HFDPCs. (A) Representative images of ALP staining in control cells and cells exposed to H2O2 (200 µM) with or without biotin (5 µg/mL), MG901-derived CFS (0.5%), or MG4237-derived CFS (2%). (B) Quantification of ALP activity. Data were expressed relative to the control. Values represent the mean ± SD of three independent experiments (n = 3). ### p < 0.001 vs. control; *** p < 0.001 vs. H2O2-treated group. Scale bar = 20 µm.
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Figure 4. Effects of MG901- and MG4237-derived CFS on intracellular ROS levels in H2O2-damaged HFDPCs. (A) Intracellular ROS generation was evaluated using the DCF-DA assay. (A) Representative fluorescence images and corresponding differential interference contrast (DIC) images were obtained from control cells and cells exposed to H2O2 (200 µM) with or without biotin (5 µg/mL), MG901-derived CFS (0.5%), or MG4237-derived CFS (2%). (B) Quantification of intracellular ROS levels based on DCF-DA fluorescence intensity. Fluorescence signals were measured at 24 h and are presented as relative values compared with the H2O2-treated group. Data represented the mean ± SD of three independent experiments (n = 3). ### p < 0.001 vs. control; *** p < 0.001 vs. H2O2-treated group. Scale bar = 50 µm.
Figure 4. Effects of MG901- and MG4237-derived CFS on intracellular ROS levels in H2O2-damaged HFDPCs. (A) Intracellular ROS generation was evaluated using the DCF-DA assay. (A) Representative fluorescence images and corresponding differential interference contrast (DIC) images were obtained from control cells and cells exposed to H2O2 (200 µM) with or without biotin (5 µg/mL), MG901-derived CFS (0.5%), or MG4237-derived CFS (2%). (B) Quantification of intracellular ROS levels based on DCF-DA fluorescence intensity. Fluorescence signals were measured at 24 h and are presented as relative values compared with the H2O2-treated group. Data represented the mean ± SD of three independent experiments (n = 3). ### p < 0.001 vs. control; *** p < 0.001 vs. H2O2-treated group. Scale bar = 50 µm.
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Figure 5. Effects of MG901- and MG4237-derived CFS on mitochondrial membrane potential in H2O2-damaged HFDPCs. Mitochondrial membrane potential was assessed using the JC-1 assay. (A) Representative fluorescence images showing JC-1 aggregates (red) and monomers (green), along with corresponding DIC images, were obtained from control cells and cells exposed to H2O2 (200 µM) with or without biotin (5 µg/mL), MG901-derived CFS (0.5%), or MG4237-derived CFS (2%). (B) Mitochondrial membrane potential was quantified using the aggregate-to-monomer fluorescence ratio. Data represented the mean ± SD of three independent experiments (n = 3). ### p < 0.001 vs. control; *** p < 0.001 vs. H2O2-treated group. Scale bar = 50 µm.
Figure 5. Effects of MG901- and MG4237-derived CFS on mitochondrial membrane potential in H2O2-damaged HFDPCs. Mitochondrial membrane potential was assessed using the JC-1 assay. (A) Representative fluorescence images showing JC-1 aggregates (red) and monomers (green), along with corresponding DIC images, were obtained from control cells and cells exposed to H2O2 (200 µM) with or without biotin (5 µg/mL), MG901-derived CFS (0.5%), or MG4237-derived CFS (2%). (B) Mitochondrial membrane potential was quantified using the aggregate-to-monomer fluorescence ratio. Data represented the mean ± SD of three independent experiments (n = 3). ### p < 0.001 vs. control; *** p < 0.001 vs. H2O2-treated group. Scale bar = 50 µm.
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Figure 6. Effects of MG901- and MG4237-derived CFS on Wnt/β-catenin signaling in H2O2-damaged HFDPCs. (A) Representative Western blot images showing β-catenin, phosphorylated GSK3β at Ser9 (p-GSK3β Ser9), total GSK3β, and β-actin expression in control cells and cells exposed to H2O2 (200 µM) with or without biotin (5 µg/mL), MG901-derived CFS (0.5%), or MG4237-derived CFS (2%). (B) β-Catenin protein levels were normalized to β-actin and expressed relative to the control. (C) Ratio of p-GSK3β (Ser9) to total GSK3β. Data represent the mean ± SD of three independent experiments (n = 3). * p < 0.05, *** p < 0.001 vs. H2O2-treated group.
Figure 6. Effects of MG901- and MG4237-derived CFS on Wnt/β-catenin signaling in H2O2-damaged HFDPCs. (A) Representative Western blot images showing β-catenin, phosphorylated GSK3β at Ser9 (p-GSK3β Ser9), total GSK3β, and β-actin expression in control cells and cells exposed to H2O2 (200 µM) with or without biotin (5 µg/mL), MG901-derived CFS (0.5%), or MG4237-derived CFS (2%). (B) β-Catenin protein levels were normalized to β-actin and expressed relative to the control. (C) Ratio of p-GSK3β (Ser9) to total GSK3β. Data represent the mean ± SD of three independent experiments (n = 3). * p < 0.05, *** p < 0.001 vs. H2O2-treated group.
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Figure 7. Schematic representation of the effects of MG901- and MG4237-derived CFS on oxidative stress–related Wnt/β-catenin signaling in HFDPCs. Oxidative stress–induced ROS disrupts β-catenin stabilization and downstream signaling, whereas MG901 and MG4237 reduce ROS levels and promote β-catenin–mediated transcriptional activity. This modulation supports dermal papilla cell function and hair growth–related responses.
Figure 7. Schematic representation of the effects of MG901- and MG4237-derived CFS on oxidative stress–related Wnt/β-catenin signaling in HFDPCs. Oxidative stress–induced ROS disrupts β-catenin stabilization and downstream signaling, whereas MG901 and MG4237 reduce ROS levels and promote β-catenin–mediated transcriptional activity. This modulation supports dermal papilla cell function and hair growth–related responses.
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Table 1. Origin and accession number of LAB strains examined in this study.
Table 1. Origin and accession number of LAB strains examined in this study.
Spp.StrainOriginNCBI Accession No.
Limosilactobacillus fermentumMG901Vaginal (Human)MN055709
Limosilactobacillus fermentumMG4237Vaginal (Human)OP102563.1
Table 2. Glucose, lactate, and acetate levels in basal medium and L. fermentum MG901 and MG4237 culture medium.
Table 2. Glucose, lactate, and acetate levels in basal medium and L. fermentum MG901 and MG4237 culture medium.
SampleGlucose (mg/L)Lactate (mg/L)Acetate (mM)
Basal medium27,022.45 ± 521.084222.96 ± 46.786.01 ± 0.02
MG901 CFS8172.66 ± 344.71 ***4199.01 ± 65.226.09 ± 0.16
MG4237 CFS7015.37 ± 342.28 ***4262.24 ± 72.047.11 ± 0.28 *
All data are presented as the mean ± SD (n = 3). Statistical significance was determined using the Kruskal–Wallis test followed by Dunnett’s multiple comparison test. * p < 0.05 and *** p < 0.001 compared the basal medium.
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MDPI and ACS Style

Jeon, C.Y.; Lee, J.Y.; Min, J.; Park, J.-Y.; Kim, M.; Yoon, W.; Choi, S.-I.; Shin, D.W. Upcycled Postbiotic Cell-Free Supernatants from Limosilactobacillus fermentum MG901 and MG4237 Alleviated Oxidative Stress-Induced Dysfunction in Human Follicle Dermal Papilla Cells. Cosmetics 2026, 13, 46. https://doi.org/10.3390/cosmetics13010046

AMA Style

Jeon CY, Lee JY, Min J, Park J-Y, Kim M, Yoon W, Choi S-I, Shin DW. Upcycled Postbiotic Cell-Free Supernatants from Limosilactobacillus fermentum MG901 and MG4237 Alleviated Oxidative Stress-Induced Dysfunction in Human Follicle Dermal Papilla Cells. Cosmetics. 2026; 13(1):46. https://doi.org/10.3390/cosmetics13010046

Chicago/Turabian Style

Jeon, Chae Young, Ji Yeon Lee, Jungwon Min, Jeong-Yong Park, Minha Kim, Wonchan Yoon, Soo-Im Choi, and Dong Wook Shin. 2026. "Upcycled Postbiotic Cell-Free Supernatants from Limosilactobacillus fermentum MG901 and MG4237 Alleviated Oxidative Stress-Induced Dysfunction in Human Follicle Dermal Papilla Cells" Cosmetics 13, no. 1: 46. https://doi.org/10.3390/cosmetics13010046

APA Style

Jeon, C. Y., Lee, J. Y., Min, J., Park, J.-Y., Kim, M., Yoon, W., Choi, S.-I., & Shin, D. W. (2026). Upcycled Postbiotic Cell-Free Supernatants from Limosilactobacillus fermentum MG901 and MG4237 Alleviated Oxidative Stress-Induced Dysfunction in Human Follicle Dermal Papilla Cells. Cosmetics, 13(1), 46. https://doi.org/10.3390/cosmetics13010046

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